A glycerol kinase (EC 2. 7. 1. 30) is an enzyme catalyzing the reaction for modifying glycerol into glycerol-3-phosphoric acid via phosphorylation reaction depending on magnesium and ATP. Since a glycerol kinase had discovered in liver by Kalckar in 1937 (see for example non-patent reference 1), it has been reported the purification of glycerol kinase from such as rat liver, pigeon liver, Candida mycoderma, Cellulomonas flavigena, Thermus flavus (see for example non-patent references 2 to 5 and patent reference 1). It has been known that the glycerol kinase exists widely in general biological organisms. Additionally, it has also been reported that gene cloning from such as human, Bacillus subtilis, Saccharomyces cerevisiae and Thermus flavus (see for example non-patent references 6 to 9). The enzyme has been studied in detail in Escherichia coli in particular. In 1967, Hayashi et al. purified the enzyme (see for example non-patent reference 10). In 1988, the cloning thereof was reported (see for example non-patent reference 11). Further, the enzyme has been studied in a wide range including research works on gene regulation and research works about the inhibition with allosteric inhibitors.
On the other hand, with regard to the industrial application of the glycerol kinase, the glycerol kinase is now used as a raw material enzyme for clinical laboratory agents. In other words, neutral fat (triglyceride) in a sample is hydrolyzed with lipase to prepare glycerol, which is then modified into glycerol-3-phosphoric acid with the enzyme. The resulting glycerol-3-phosphoric acid is used for assaying blood neutral lipid by calorimetric analysis using an oxidase of glycerol-3-phosphoric acid and ultraviolet absorptiometry using dehydrogenase of glycerol-3-phosphoric acid.
Recent clinical laboratory agents for biochemical tests have mainly been laboratory agents at solution state. Therefore, it is demanded that such laboratory agents in liquid should have high stability in addition to the characteristic features (high reactivity with substrates, strict substrate specificity, etc.) traditionally demanded for enzymes. Various characteristic features contributing to the stability of test agents in liquid can be suggested. Generally, preservative is added so as to enable long-term storage of test agents in liquid. Since such preservative may sometimes make enzymes unstable, high resistance against preservative is one of desirable enzyme properties for test agents.
It has been believed so far that enzymes which have high thermal stability show high stability in test agents in liquid. Therefore, a glycerol kinase derived from thermophilic bacteria such as Bacillus stearothermophilus and Thermus flavus has been commonly used. However, such glycerol kinase has a problem of low resistance against preservative.
Patent reference 1: JP-A-56-121484
Non-patent reference 1: H. Kalckar, eds., “Enzymologia”, Vol.2, p. 47, 1937
Non-patent reference 2: C. Bublitz, et al., “J. Biol. Chem.”, Vol. 211, p. 951, 1954
Non-patent reference 3: E. P. Kennedy, “Methods Enzymol.”, Vol. 5, p. 476, 1962
Non-patent reference 4: H. U. Bergmeyer, et al., “Biochem.”, Vol. 333, p. 471, 1961
Non-patent reference 5: H. S. Huang, et al., “J. Ferment. Bioeng.”, No. 83, p. 328, 1997
Non-patent reference 6: C. A. Sargent, et al., “Hum. Mol. Genet.”, Vol. 3, p. 1317, 1994
Non-patent reference 7: C. Holmberg, et al., “J. Gen. Microbiol.”, Vol. 136, p. 2367, 1990
Non-patent reference 8: P. Pavlik, et al., “Curr. Genet.”, Vol. 24, p. 21, 1993
Non-patent reference 9: H. S. Huang, et al., “Biochim. Biophys. Acta”, Vol. 1382, p. 186, 1998
Non-patent reference 10: S. Hayashi, et al., “J. Biol. Chem.”, Vol. 242, p. 1030, 1967
Non-patent reference 11: D. W. Pettigrew, et al., “J. Biol. Chem.”, Vol. 263, p. 135, 1988